RACGAP1 promotes lung cancer cell proliferation through the PI3K/AKT signaling pathway

We aimed to investigate the expression and clinic significance of Rac GTPase Activating Protein 1 (RACGAP1) in human lung adenocarcinoma (LUAD). Online database analysis revealed a significant increase in RACGAP1 mRNA expression among 26 types of tumor tissues, including LUAD tissues. Online database and tissue microarray analyses indicated that RACGAP1 expression was significantly upregulated in LUAD tissues. Genetic variation analysis identified four different genetic variations of RACGAPs in LUAD. Moreover, online database analysis showed that RACGAP1 upregulation was correlated with shorter survival in patients with LUAD. After silencing RACGAP1 expression in A549 cells using siRNA and assessing its protein levels via Western blotting, we found that RACGAP1 knockdown inhibited cell growth and induced apoptosis determined using the Cell Counting Kit-8 assay, colony formation assay, and flow cytometry. Mechanistically, western blot analysis indicated that Bax expression increased, whereas Bcl-2 expression decreased. Moreover, RACGAP1 knockdown attenuated PI3K/AKT pathway activation in lung cancer cells. Taken together, our findings showed that RACGAP1 was overexpressed in LUAD tissues and played an important role in lung cancer by increasing cell growth through the PI3K/AKT signaling pathway. This study suggests recommends evaluating RACGAP1 in clinical settings as a novel biomarker and potential therapeutic target for lung cancer.

adenocarcinoma patients are summarized in Table 1.RACGAP1 expression was significantly greater in tumor tissues than in adjacent normal tissues.A significant difference in RACGAP1 staining pattern was observed between the two groups (Fig. 2c, P < 0.05).The aforementioned findings revealed that RACGAP1 was upregulated in LUAD at both the mRNA and protein levels.

Relationship between RACGAP1 expression and clinicopathological parameters of LUAD patients
To investigate the correlation between RACGAP1 expression and clinicopathological parameters of patients, the UALCAN database was used to obtain information regarding age, sex, smoking habits, disease stage, nodal metastasis, and p53 mutants, after which their relationship with RACGAP1 levels were determined.Interestingly,

Upregulation of RACGAP1 was correlated with shorter survival in LUAD patients
The current study further determined whether RACGAP1 was correlated with LUAD patient survival through both GEPIA and Kaplan-Meier survival curves.As shown in Fig. 5a, GEPIA indicated that increased RACGAP1 expression was correlated with decreased overall survival in LUAD patients (P = 1.70E-04).Consistently, analyses using the Kaplan-Meier Plotter revealed that increased RACGAP1 expression was significantly correlated with decreased overall survival (OS; Fig. 5b; hazard ratio [HR] 1.58, log-rank P = 2.0E−12), progression-free survival (PFS; Fig. 5c; HR 1.48, log-rank P = 5.5E−05), and post-progression survival (PPS; Fig. 5d; HR 1.5, log-rank P = 1.7E−03).These findings suggest the potential role of RACGAP1 as a biomarker for predicting prognosis.

RACGAP1 knockdown inhibits A549 cell growth
To explore the biological function of RACGAP1 on LUAD cell growth, three independent siRNAs targeting RAC-GAP1 were designed, and knockdown efficiencies were determined using Western blotting.Notably, Western blot analyses revealed that RACGAP1 expression was significantly downregulated at the protein level in A549 cells with all three siRNAs (Fig. 6a).Next, we used transfected with A549 cells to evaluate the effects of RACGAP1   www.nature.com/scientificreports/ on cellular functions.As shown in Fig. 6b, RACGAP1 knockdown reduced the viability of A549 cells.Moreover, RACGAP1 knockdown inhibited the formation of colonies (Fig. 6c).To further elucidate the possible mechanism underlying cellular growth inhibition induced by RACGAP1 knockdown, the cell cycle assay was performed using flow cytometry.As shown in Fig. 7, RACGAP1 knockdown markedly increased the percentage of cells in the G0/G1 phase but decreased the percentage of cells in the S phase (Fig. 7a), as well as significantly downregulated the expression of G0/G1-related proteins CyclinD1 and CDK4 in A549 cells, excepted for si-RACGAP1-3 in Cyclin D1 and si-RACGAP1 inCDK4 (Fig. 7b).These results suggest that RACGAP1 knockdown may inhibit proliferation by inducing G0/G1 phase arrest.

RACGAP1 knockdown induces apoptosis of A549 cells
To investigate the effects of RACGAP1 on cell apoptosis, Annexin V-647 and PI were used to stain apoptotic cells.

Discussion
Considering its malignant nature, lung cancer has been associated with high morbidity and mortality rates 1,3 .
Diagnosis at an early stage, which can dramatically increase cure rates, is important for LUAD treatment.Owing to the poor effects of molecular targeted therapy in advanced-stage lung cancer, the prognosis of LUAD patients remains poor.Therefore, further studies on novel potential biomarker for early diagnosis and prognostic prediction are urgent needed for lung cancer patients.For this purpose, the current study analyzed multiple databases, with our results showing that RACGAP1 mRNA expression was upregulated in multiple tumor tissues (including lung, liver, and colorectal cancers among others) compared to normal adjacent tissues.These findings confirm that RACGAP1 expression was widely upregulated in several types of cancers, including stomach 15 , breast 16,17 and liver cancers 20 , suggesting that RACGAP1 upregulation might be a common occurrence and plays an essential role in the development and progression of cancers.Consistent with our finding on mRNA levels, we also confirmed that RACGAP1 protein expression was significantly upregulated in LUAD tissues compared to normal lung tissues.However, more clinic samples need to be analyzed to further verify RACGAP1 expression in future studies.Furthermore, we used the cBioPortal database to analyze genetic variations of RACGAP1 in LUAD, from which we identified four different genetic variations of RACGAP1, including missense mutation, truncating mutation, amplification, and deep deletion.These results indicate that RACGAP1 could potentially play a role of in LUAD diagnosis.Moreover, we analyzed the relationship between RACGAP1 expression and clinical pathological parameters of LUAD patients.Notably, our findings showed that RACGAP1 was significantly correlated with male sex, smoking, stage, nodal metastasis, and TP53 mutant status in LUAD patients, a finding consistent with those presented in previous study on breast and ovarian cancers 24,25 .Our survival analysis based on the Kaplan-Meier Plotter online database and GEPIA database revealed that increased RACGAP1 mRNA expression was associated with poor OS and PFS.The current study found that silencing of RACGAP1 expression in A549 cells using three different siRNAs inhibited  cell growth as evidenced by decreased cell viability and colony number.Moreover, our analysis revealed that RACGAP1 knockdown blocked G0/G1 progression downregulated the expression of cyclinD1 and CDK4 in A549 cells.Furthermore, the anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax, which both belong to the Bcl-2 family, have been found to be critical regulators of this pathway 26 .Notably, we observed that RACGAP1 knockdown increased A549 cell apoptosis.Our analysis of protein expression levels showed that RACGAP1 knockdown downregulated Bcl-2 but upregulated Bax.These findings demonstrated that disruptions in the balance between proliferation and apoptosis in the development of LUAD could be attributed to RACGAP1 overexpression.
The PI3K/AKT signaling pathway is a pivotal cellular signaling cascade that exerts profound regulatory influence over fundamental cellular processes including proliferation, growth, survival, and metabolism 27 .Activated AKT orchestrates cellular proliferation and growth through multiple pathways.It directly influences cell cycle regulatory proteins, facilitating progression through S and G2/M phases.Additionally, AKT modulates protein synthesis and cell size to promote cellular growth.Furthermore, AKT functions as a crucial regulator of cell survival and apoptosis inhibition.Through modulation of signaling molecules such as the Bcl-2 family and  www.nature.com/scientificreports/caspases, AKT suppresses apoptosis and promotes cell viability, thereby maintaining cellular homeostasis 28 .Therefore, we found that RACGAP1 knockdown decreased the protein expression of PI3K and p-AKT, it had only minor effects on the total expression of AKT.The suggests that suppression of PI3K/AKT pathway might be among the underlying mechanisms behind the suppression of LUAD cell growth by silencing of RACGAP1.In summary, the current study analyzed RACGAP1 mRNA and protein expression in multiple cancers, including lung cancer, and determined their correlation with prognosis and clinicopathological features.Overall, we found that RACGAP1 knockdown exerted anti-tumor activity in vitro, indicating that RACGAP1 might be involved in lung cancer development.Nonetheless, the underlying regulatory mechanisms still need more explored.

Bioinformatics analysis
GEPIA (http:// gepia.cancer-pku.cn/) was performed to analyze the expression levels of RACGAP1 in different types of cancers and overall survival analysis of RACGAP1 proteins from TCGA and Genotype-Tissue Expression, especially lung cancer, including LUAD and LUSC, and paired normal tissues.Changes in RACGAP1 mRNA levels in LUAD/LUSC and healthy tissues were further verified using the UALCAN online database.The protein expression of RACGAP1 in LUAD samples was analyzed through HPA and UALCAN databases.

Immunohistochemistry-based TMA
The lung cancer samples and matched noncancerous lung tissues (cat no.HLugA150CS03), containing 75 pairs of lung adenocarcinoma tissues and adjacent noncancerous lung tissues, were used for TMA construction.TMAs were created through a contract service at Shanghai OUTDO Biotech, China.The RACGAP1 antibody (1:3000; cat no.13739-1-AP; Proteintech Group, Wuhan, China) was used to determine protein expression levels.Human tissues were stained using the EliVision Plus Kit (Maixin, China) according to the manufacturer's instructions.The RACGAP1 immunostaining score was calculated as the sum of the staining intensity score and positive staining cell rate score.The staining intensity was scored as follows: no staining: 0, weak staining: 1, moderate staining: 2, and strong staining: 3. The positive staining cell rate was scored as follows: 0% to 5%: 0, 5% to 25%: 1, 26% to 50%: 2, 51% to 75%: 3, and > 75%: 4. The Wilcoxon test (raw scores) was applied to determine the significance of RACGAP1 staining in primary lung tumors relative to the matched adjacent tumoral tissues 29,30 .

Genetic alteration analysis
The cBioPortal database was used to obtain information on the genetic variations of RACGAP1 in 2068 cases retrieved from 7 studies (35 cases from MSKCC; 230 cases from TCGA pub; 566 cases from TCGA, PanCancer; 302 cases from OncoSG, 2020; 516 cases from TCGA; 183 cases from Broad; and 0 cases from TSP).

Clinicopathologic features
The UALCAN database was also used to investigate the relationship between RACGAP1 expression levels and different clinicopathological parameters, which included age, male sex, smoking, stage, nodal metastasis, and TP53 mutant status (summarized in Table 1).

Survival analysis
The Kaplan-Meier plotter (www.kmplot.com), containing gene expression data and survival information of patients with LUAD, was queried to analyze the association between RACGAP1 mRNA expression and survival of patients with LUAD, including OS, PFS and PPS.The expression of RACGAP1 among the samples was divided into high or low groups according to the median expression, and the association of RACGAP1 expression with the survival of patients with LUAD was analyzed using the log-rank test method.

Cell line and culture
The lung carcinoma cell line A549 was acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).Cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), penicillin (100,000 U/L), and streptomycin (100 mg/L) at 37 °C in a 5% CO 2 atmosphere.The cell lines were tested and authenticated.

Cell transfection
Three different siRNAs, namely anti-RACGAP1 (si-RACGAP1-1/-2/-3) and control siRNA (si-Ctrl), were purchased from Ribobio (Guangzhou, Guangdong, China).Cells were transfected with siRNAs or si-Ctrl at a concentration of 100 nM using Lipofectamine RNAiMax (Thermo Fisher Scientific) for 6-8 h according to manufacturer's instructions.The cells were then cultured with complete medium at 37℃ in a humidified atmosphere with 5% CO 2 for the indicated time points prior to use for experiments.

Figure 2 .
Figure 2. RACGAP1 protein expression in lung cancer tissues and normal tissues.(a) Immunohistochemical staining of RACGAP1 in LUAD tissues and normal lung tissues from the HPA database.(b) RACGAP1 protein expression in LUAD tissues and normal lung tissues from CPTAC samples.(c) RACGAP1 protein levels were determined via IHC using tissue microarrays in 75 pairs of lung adenocarcinoma and adjacent noncancerous lung tissues.The lower right panels show representative images captured at × 4 and × 20 magnification.Each datapoint in the plot represents one tissue sample.Error bars represent the median ± range.T, lung cancer tissue; N, noncancerous lung tissue.*P < 0.05 vs. normal tissues.

Figure 3 .
Figure 3. Analysis of RACGAP1 alterations in LUAD.(a) CNA and mutation frequency data for RACGAP1 in LUAD were analyzed through the cBioPortal website.(b) OncoPrint visual summary of alterations on a query of RACGAP1.Variations in frequency included missense mutation (green), truncating mutation (gray), amplification (red), and deep deletions (blue).

Figure 5 .
Figure 5. Correlation between RACGAP1 expression and survival of LUAD patients.(a) The correlation between RACGAP1 expression and OS of LUAD patients was analyzed using GEPIA.(b-d) The correlation between RACGAP1 expression and OS, PFS, or PPS of LUAD patients was analyzed through the Kaplan-Meier Plotter.OS, overall survival; PFS, free-progression survival; PPS, post-progression survival.

Figure 6 .
Figure 6.RACGAP1 knockdown inhibited cell growth in A549 cells.A549 cells were transfected with si-Ctrl or three independent siRNAs for si-RACGAP1.(a) Protein levels of RACGAP1 were determined using Western blot analysis.Representative images of RACGAP1 and β-actin are shown and quantitated using ImageLab software.β-actin was used as an internal control.*P < 0.05 vs. si-Ctrl.(b) Cell viability was determined using the CCK-8 assay.Data were normalized to the viability on day 1 and are represented as fold change.*P < 0.05 vs. si-Ctrl.(c) The colony formation assay was used to determine cell survival.After obtaining the images, colony formation was calculated and normalized to the survival of control cells.*P < 0.05 vs. si-Ctrl.

Figure 7 .
Figure 7. RACGAP1 knockdown inhibited cell proliferation in A549 cells.A549 cells were transfected with si-Ctrl or three independent siRNAs for si-RACGAP1.(a) Cell cycle progression was determined through PI staining and FACS analysis.Representative plots and percentage of cells at different stages (G0/G1, G2/M, and S phases) were presented.*P < 0.05 vs. si-Ctrl.(b) Protein levels of CDK4 and CyclinD1 after RACGAP1 knockdown were determined using Western blot analysis.Representative images are shown and quantitated using ImageLab software.β-actin was used as an internal control.*P < 0.05 vs. si-Ctrl.

Figure 8 .
Figure 8. RACGAP1 knockdown induced cell apoptosis in A549 cells.A549 cells were transfected with si-Ctrl or three independent siRNAs for si-RACGAP1.(a) Cell apoptosis was determined using Annexin V/PI staining and FACS analysis.Representative plots and percentage of apoptotic cells are presented.*P < 0.05 vs. si-Ctrl.(b) Protein levels of Bax and Bcl-2 after RACGAP1 knockdown were determined using Western blot analysis.Representative images are shown and quantitated using ImageLab software.β-actin was used as an internal control.*P < 0.05 vs. si-Ctrl.

Figure 9 .
Figure 9. RACGAP1 knockdown inhibited the PI3K/AKT signaling pathway in A549 cells.A549 cells were transfected with si-Ctrl or si-RACGAP1.Protein levels of PI3K, p-AKT, and AKT after RACGAP1 knockdown were determined using Western blot analysis.Representative images are shown and quantitated using ImageLab software.GAPDH was used as an internal control.*P < 0.05 vs. si-Ctrl.

Table 2 .
Clinicopathological features of patients.